Mouse Perturbation Core D is central the mission of this U19 Program because it will execute CRISPR/Cas9 forward genetic screens in mice to enable U19 investigators to identify novel regulators of innate and adaptive immune responses to acute infection in mouse models. Core D will make an innovative technology for conducting Cas9/CRISPR-mediated genetic screens easily accessible to all U19 investigators. Core D will assist U19 investigators with screen design; generate and validate bone marrow chimeric mice carrying curated pools of sgRNA; infect these bone marrow chimeras or recipients of T cells carrying sgRNAs from these bone marrow chimeras with virus for U19 investigators; and assist U19 investigators with validation of candidate regulators in acute viral infection models. Core D has expertise with all of these procedures and demonstrated success with using these approaches to conduct forward genetic screens in vivo. To achieve these goals, the Specific Aims of Core D are: 1) To generate and validate bone marrow chimeric mice carrying sgRNA libraries. Core D will provide advice on screen design and then transduce the appropriate Cas9-expressing hematopoietic progenitor cells with lentivirus carrying curated libraries of sgRNAs generated by Core C; 2) To infect bone marrow chimeric mice or recipients of T cells from these mice with virus. Core D will infect bone marrow chimeras or recipients of transduced T cells from bone marrow chimeras for in vivo screens (using LCMV Armstrong), and for in vivo validation studies of candidate regulators of innate and adaptive immune responses to acute infection (using LCMV Armstrong or influenza virus). Core D also will provide transduced DCs from bone marrow chimeras for ex vivo screens to identify regulators of DC interactions with pathogens, pathogen components and T cells; 3) To generate, maintain and validate TCR transgenic and immune-lineage-specific Cas9 expressing mice, and LCMV Armstrong and influenza viral stocks. Core D will serve as a repository for Cas9-expressing mouse strains and viral stocks. This centralized approach will standardize the set of procedures needed to conduct forward genetic screens in vivo. Provision of this novel technology by a core provides an efficient and cost-effective means to conduct these in vivo screens, to ensure uniformity and success in use of these approaches by U19 investigators, and to facilitate comparisons of data across the U19 Program. Core D will work closely with investigators in Project 1 and 2 to help them conduct in vivo screens and validate candidate regulators, and with Core C for lentiviral pools of sgRNA for the screens, Core A to facilitate communication and administrative oversight of Core D activities, and Core B through the Project investigators for computational analyses of single cell data from immune populations.